The intermediate filament protein peripherin is the specific interaction partner of mouse BPAG1-n (dystonin) in neurons.

Leung CL, Sun D, Liem RK - J. Cell Biol. (1999)

Bottom Line:
Kouklis, D.W.Fuchs. 1996.Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.

Affiliation: Departments of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York 10032, USA.

ABSTRACTThe dystonia musculorum (dt) mouse suffers from severe degeneration of primary sensory neurons. The mutated gene product is named dystonin and is identical to the neuronal isoform of bullous pemphigoid antigen 1 (BPAG1-n). BPAG1-n contains an actin-binding domain at its NH2 terminus and a putative intermediate filament-binding domain at its COOH terminus. Because the degenerating sensory neurons of dt mice display abnormal accumulations of intermediate filaments in the axons, BPAG1-n has been postulated to organize the neuronal cytoskeleton by interacting with both the neurofilament triplet proteins (NFTPs) and microfilaments. In this paper we show by a variety of methods that the COOH-terminal tail domain of mouse BPAG1 interacts specifically with peripherin, but in contrast to a previous study (Yang, Y., J. Dowling, Q.C. Yu, P. Kouklis, D.W. Cleveland, and E. Fuchs. 1996. Cell. 86:655-665), mouse BPAG1 fails to associate with full-length NFTPs. The tail domains interfered with the association of the NFTPs with BPAG1. In dt mice, peripherin is present in axonal swellings of degenerating sensory neurons in the dorsal root ganglia and is downregulated even in other neural regions, which have no obvious signs of pathology. Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.

Figure 2: Analysis of mBPAG-C1 with peripherin, α-internexin, and vimentin filament networks in transient transfection studies. SW13.cl.2Vim− cells from transient transfections of pRSVi-peripherin (C), pRSVi-α-internexin (F), or pRSVi-vimentin (I), and cotransfections of pcDNA-FLAG-mBPAG-C1 with (A and B) pRSVi-peripherin, (D and E) pRSVi-α-internexin, or (G and H) pRSVi-vimentin were double-labeled with mouse anti-FLAG M2 mAb (A, D, and G) and rabbit polyclonal antibodies against peripherin (B and C), α-internexin (E and F), or vimentin (H and I). mBPAG-C1 associated with the filament network of peripherin, but disrupted the normal filament network of α-internexin. The perturbed α-internexin filament bundles were decorated with mBPAG-C1. The expressed mBPAG-C1 also appeared to affect the morphology of the vimentin filament network. Bar, 10 μm.

Mentions:
The results of the two-hybrid experiments prompted us to repeat some of the transient transfection assays performed by Yang et al. (1996) on mBPAG-C1 and NFTPs. In addition, we wanted to confirm the interactions of mBPAG-C1 with α-internexin and peripherin. To facilitate the detection of mBPAG-C1 in the transient transfection assays, the NH2 terminus of mBPAG-C1 was fused to a FLAG epitope tag, which also provided the translational start codon (Fig. 1). All transfections were performed in a human adrenal carcinoma cell line, SW13.cl.2Vim−, because it did not contain any cytoplasmic IFs that might interact with mBPAG-C1. Cotransfections of mBPAG-C1 with peripherin showed colocalization of mBPAG-C1 with peripherin filament networks in transiently transfected cells (Fig. 2, A and B). In contrast, mBPAG-C1 appeared to disrupt the normal filament formation of α-internexin. α-Internexin has been shown previously to self-assemble into normal filamentous networks in transiently transfected SW13.cl.2Vim− cells (Fig. 2 F; Ching and Liem, 1993). In the presence of mBPAG-C1, the filaments formed by α-internexin became bundled and these bundles were decorated with mBPAG-C1 (Fig. 2, D and E). In addition, the punctate background staining patterns observed with both antibodies suggest that interaction with mBPAG-C1 prevented some α-internexin molecules from self-assembly into filaments. Although mBPAG-C1 did not colocalize with vimentin filaments in transfected cells, it seemed to have some influence on the morphology of the assembled vimentin filament network (Fig. 2, G–I). Since none of the NFTP subunits can self-assemble into filamentous networks (Ching and Liem, 1993; Lee et al., 1993), transient transfections of mBPAG-C1 with NF-L and NF-M or with NF-L and NF-H were performed in order to detect potential interactions between BPAG1-n and polymerized NFs. In contrast to a previous report (Yang et al., 1996), we did not observe any colocalization between mBPAG-C1 and the filament networks formed by NF-L and NF-M or by NF-L and NF-H. Instead, the immunostaining pattern of mBPAG-C1 was diffuse in the presence of these filament networks in the transfected SW13.cl.2Vim− cells (Fig. 3). To verify that these patterns were indeed distinct, we performed confocal microscopy on the cells cotransfected with mBPAG-C1, NF-L, and NF-H. As shown in Fig. 3, C and D, the filamentous staining pattern observed for NF-L/NF-H filaments did not colocalize with the diffuse mBPAG-C1 staining pattern.

Figure 2: Analysis of mBPAG-C1 with peripherin, α-internexin, and vimentin filament networks in transient transfection studies. SW13.cl.2Vim− cells from transient transfections of pRSVi-peripherin (C), pRSVi-α-internexin (F), or pRSVi-vimentin (I), and cotransfections of pcDNA-FLAG-mBPAG-C1 with (A and B) pRSVi-peripherin, (D and E) pRSVi-α-internexin, or (G and H) pRSVi-vimentin were double-labeled with mouse anti-FLAG M2 mAb (A, D, and G) and rabbit polyclonal antibodies against peripherin (B and C), α-internexin (E and F), or vimentin (H and I). mBPAG-C1 associated with the filament network of peripherin, but disrupted the normal filament network of α-internexin. The perturbed α-internexin filament bundles were decorated with mBPAG-C1. The expressed mBPAG-C1 also appeared to affect the morphology of the vimentin filament network. Bar, 10 μm.

Mentions:
The results of the two-hybrid experiments prompted us to repeat some of the transient transfection assays performed by Yang et al. (1996) on mBPAG-C1 and NFTPs. In addition, we wanted to confirm the interactions of mBPAG-C1 with α-internexin and peripherin. To facilitate the detection of mBPAG-C1 in the transient transfection assays, the NH2 terminus of mBPAG-C1 was fused to a FLAG epitope tag, which also provided the translational start codon (Fig. 1). All transfections were performed in a human adrenal carcinoma cell line, SW13.cl.2Vim−, because it did not contain any cytoplasmic IFs that might interact with mBPAG-C1. Cotransfections of mBPAG-C1 with peripherin showed colocalization of mBPAG-C1 with peripherin filament networks in transiently transfected cells (Fig. 2, A and B). In contrast, mBPAG-C1 appeared to disrupt the normal filament formation of α-internexin. α-Internexin has been shown previously to self-assemble into normal filamentous networks in transiently transfected SW13.cl.2Vim− cells (Fig. 2 F; Ching and Liem, 1993). In the presence of mBPAG-C1, the filaments formed by α-internexin became bundled and these bundles were decorated with mBPAG-C1 (Fig. 2, D and E). In addition, the punctate background staining patterns observed with both antibodies suggest that interaction with mBPAG-C1 prevented some α-internexin molecules from self-assembly into filaments. Although mBPAG-C1 did not colocalize with vimentin filaments in transfected cells, it seemed to have some influence on the morphology of the assembled vimentin filament network (Fig. 2, G–I). Since none of the NFTP subunits can self-assemble into filamentous networks (Ching and Liem, 1993; Lee et al., 1993), transient transfections of mBPAG-C1 with NF-L and NF-M or with NF-L and NF-H were performed in order to detect potential interactions between BPAG1-n and polymerized NFs. In contrast to a previous report (Yang et al., 1996), we did not observe any colocalization between mBPAG-C1 and the filament networks formed by NF-L and NF-M or by NF-L and NF-H. Instead, the immunostaining pattern of mBPAG-C1 was diffuse in the presence of these filament networks in the transfected SW13.cl.2Vim− cells (Fig. 3). To verify that these patterns were indeed distinct, we performed confocal microscopy on the cells cotransfected with mBPAG-C1, NF-L, and NF-H. As shown in Fig. 3, C and D, the filamentous staining pattern observed for NF-L/NF-H filaments did not colocalize with the diffuse mBPAG-C1 staining pattern.

Bottom Line:
Kouklis, D.W.Fuchs. 1996.Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.

Affiliation:
Departments of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York 10032, USA.

ABSTRACTThe dystonia musculorum (dt) mouse suffers from severe degeneration of primary sensory neurons. The mutated gene product is named dystonin and is identical to the neuronal isoform of bullous pemphigoid antigen 1 (BPAG1-n). BPAG1-n contains an actin-binding domain at its NH2 terminus and a putative intermediate filament-binding domain at its COOH terminus. Because the degenerating sensory neurons of dt mice display abnormal accumulations of intermediate filaments in the axons, BPAG1-n has been postulated to organize the neuronal cytoskeleton by interacting with both the neurofilament triplet proteins (NFTPs) and microfilaments. In this paper we show by a variety of methods that the COOH-terminal tail domain of mouse BPAG1 interacts specifically with peripherin, but in contrast to a previous study (Yang, Y., J. Dowling, Q.C. Yu, P. Kouklis, D.W. Cleveland, and E. Fuchs. 1996. Cell. 86:655-665), mouse BPAG1 fails to associate with full-length NFTPs. The tail domains interfered with the association of the NFTPs with BPAG1. In dt mice, peripherin is present in axonal swellings of degenerating sensory neurons in the dorsal root ganglia and is downregulated even in other neural regions, which have no obvious signs of pathology. Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.